PURPOSE: To identify the medical and biological significance of the novel non-neuronal cholinergic system of the upper digestive tract, and elucidate the effects of pure nicotine (Nic) vs. tobacco products on nicotinic and muscarinic acetylcholine receptors (nAChR and mAChR) expressed by oral and esophageal keratinocytes (KC). BACKGROUND: ACh regulates vital functions of KC including proliferation, adhesion, migration, differentiation and apoptosis. The alpha3, alpha5, alpha7, alpha9, beta2, and beta4 nAChR subunits, the m2, m3, m4 and m5 mAChR subtypes, as well as the synthesizing enzyme choline acetyltransferase and the degrading enzyme acetylcholinesterase are expressed and function in human oral KC, and may target KC for direct effects of Nic. PRELIMINARY OBSERVATIONS: Concomitant emergence of different ACh receptors in the course of KC differentiation allows ACh to exhibit diverse and temporally regulation of the developing mucosal epithelium. A stimulatory effect of ACh on Ca2+ influx, mediated by nAChR, balances an inhibitory effect, mediated by mAChR, and simultaneous activation of both receptors produces a kind of a yin yang regulatory balance in KC. HYPOTHESES: 1) Each ACh receptor expressed by oral KC regulates uniquely the cell cycle progression so that the differentiation-determined changes in the repertoire of ACh receptors in a single keratinocyte can diversify the biological effects of ACh. 2) Chronic exposure to Nic disturbs the dynamic equilibrium between the nicotinic and the muscarinic pathways of keratinocyte control by ACh, leading to an aberrant cell cycle. SPECIFIC AIMS: 1) Identify contribution of each ACh receptor to the cell cycle progression using subtype-selective agonists and antagonists, antisence oligonucleotides, and knockout mice. 2) Identify long-term effects of Nic vs. aqueous extract of smokeless tobacco vs. environmental tobacco smoke on expression/function of keratinocyte ACh receptors and the Ca2+ pathways subserving ACh signaling in KC. METHODOLOGY: A combination of molecular biological (RT-PCR, real-time TaqMan PCR, Northern blots), immunological (immunohistochemistry, Western blots, ELISA), pharmacological and biological assays will be employed to quantitate ACh receptor-mediated changes of the cell cycle progression (Aim #1) as well as effects of Nic and tobacco products on the structure and function of keratinocyte ACh receptors (Aim #2). SIGNIFICANCE: The role of non-neuronal ACh in oral cell biology and pathology will be elucidated, which will help explain some of the mechanisms of deleterious effects of tobacco products in the upper digestive tract.